PROJECT ABSTRACT Targeting novel cellular processes is critical to maintaining a fresh antibiotic development pipeline. In that regard, we hypothesize that the essential Staphylococcus aureus protein, RnpA, is a unique and druggable antimicrobial target. We also predict that successful RnpA inhibitor development campaigns will provide new classes of broad spectrum antibiotics that can be used for the therapeutic intervention of S. aureus, other Gram-positive organisms and Gram-negative pathogens of immediate and emerging healthcare concern. Accordingly, we have assembled a multi-disciplinary team of microbiologists, biochemists, chemists, computational and structural biologists to launch a two-pronged approach to determine whether physiologically promising RnpA inhibitors can be reasonably achieved. Specifically, we will use a whole cell RnpA inhibitor enrichment assay combined with a validated enzyme target based approach (Aim 1) and fragment-based screening approach (Aim 2) to arrive at RnpA inhibitors. A benefit of this program is that it allows integration of structural and computational data for rational hit optimization regardless of their identification route and will be guided by our recently solved S. aureus RnpA X-ray crystal structure. While the approaches used are cutting-edge, the true innovation of this project may reside in the target itself. RnpA forms two holoenzymes, both of which mediate distinct and putatively independently essential cellular functions, RNA degradation and ptRNA maturation. Consequently, targeting RnpA potentially allows three routes of identifying therapeutically promising chemical classes of inhibitors and project success, including those that inhibit the protein's modulation of: 1. mRNA decay, 2. ptRNA processing or 3. Both processes.